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Yorodumi- PDB-8gkh: Structure of the Spizellomyces punctatus Fanzor (SpuFz) in comple... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8gkh | ||||||
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Title | Structure of the Spizellomyces punctatus Fanzor (SpuFz) in complex with omega RNA and target DNA | ||||||
Components |
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Keywords | RNA BINDING PROTEIN/RNA/DNA / Fanzor / Eukaryotic / RNA-guided / nuclease / Gene editing / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA-DNA complex | ||||||
Function / homology | Function and homology information carbohydrate transmembrane transporter activity / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing Similarity search - Function | ||||||
Biological species | Methanosarcina mazei (archaea) Spizellomyces punctatus (fungus) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å | ||||||
Authors | Xu, P. / Saito, M. / Zhang, F. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nature / Year: 2023 Title: Fanzor is a eukaryotic programmable RNA-guided endonuclease. Authors: Makoto Saito / Peiyu Xu / Guilhem Faure / Samantha Maguire / Soumya Kannan / Han Altae-Tran / Sam Vo / AnAn Desimone / Rhiannon K Macrae / Feng Zhang / Abstract: RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both ...RNA-guided systems, which use complementarity between a guide RNA and target nucleic acid sequences for recognition of genetic elements, have a central role in biological processes in both prokaryotes and eukaryotes. For example, the prokaryotic CRISPR-Cas systems provide adaptive immunity for bacteria and archaea against foreign genetic elements. Cas effectors such as Cas9 and Cas12 perform guide-RNA-dependent DNA cleavage. Although a few eukaryotic RNA-guided systems have been studied, including RNA interference and ribosomal RNA modification, it remains unclear whether eukaryotes have RNA-guided endonucleases. Recently, a new class of prokaryotic RNA-guided systems (termed OMEGA) was reported. The OMEGA effector TnpB is the putative ancestor of Cas12 and has RNA-guided endonuclease activity. TnpB may also be the ancestor of the eukaryotic transposon-encoded Fanzor (Fz) proteins, raising the possibility that eukaryotes are also equipped with CRISPR-Cas or OMEGA-like programmable RNA-guided endonucleases. Here we report the biochemical characterization of Fz, showing that it is an RNA-guided DNA endonuclease. We also show that Fz can be reprogrammed for human genome engineering applications. Finally, we resolve the structure of Spizellomyces punctatus Fz at 2.7 Å using cryogenic electron microscopy, showing the conservation of core regions among Fz, TnpB and Cas12, despite diverse cognate RNA structures. Our results show that Fz is a eukaryotic OMEGA system, demonstrating that RNA-guided endonucleases are present in all three domains of life. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8gkh.cif.gz | 219.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8gkh.ent.gz | 157 KB | Display | PDB format |
PDBx/mmJSON format | 8gkh.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8gkh_validation.pdf.gz | 1 MB | Display | wwPDB validaton report |
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Full document | 8gkh_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | 8gkh_validation.xml.gz | 37.3 KB | Display | |
Data in CIF | 8gkh_validation.cif.gz | 55.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/gk/8gkh ftp://data.pdbj.org/pub/pdb/validation_reports/gk/8gkh | HTTPS FTP |
-Related structure data
Related structure data | 40184MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-DNA chain , 2 types, 2 molecules NT
#1: DNA chain | Mass: 4594.986 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
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#3: DNA chain | Mass: 10737.919 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Protein / RNA chain , 2 types, 2 molecules PW
#2: Protein | Mass: 118029.922 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Methanosarcina mazei (archaea), (gene. exp.) Spizellomyces punctatus (fungus) Gene: malE / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A0F8NYV9, UniProt: A0A0L0H5U9 |
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#4: RNA chain | Mass: 25050.807 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Spizellomyces punctatus (fungus) / Production host: Saccharomyces cerevisiae (brewer's yeast) |
-Non-polymers , 3 types, 6 molecules
#5: Chemical | #6: Chemical | ChemComp-ZN / | #7: Water | ChemComp-HOH / | |
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-Details
Has ligand of interest | N |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: SpuFz-wRNA-tgDNA complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT |
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Source (natural) | Organism: Spizellomyces punctatus (fungus) |
Source (recombinant) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE-PROPANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2600 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm |
Image recording | Electron dose: 51.41 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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3D reconstruction | Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 501142 / Symmetry type: POINT |